Pyrrole compounds for detecting the presence of hydrolytic analytes

ABSTRACT

A novel compound useful in detecting leukocytes, esterase and protease in a test sample. The compound has the structure ##STR1## in which: A is an acid residue, R is lower alkyl, aryl, carboxyl, carboxyl ester, amido or cyano, R* is H or lower alkyl, and X is O, S, or NR&#39;, in which R&#39; is H, lower alkyl or aryl.

CONTENTS

1. Introduction

2. Background of the Invention

3. Summary of the Invention

4. Definitions

4.1 Acid Residue

4.2 Aryl

4.3 Lower alkyl

5. Detailed Description of the Invention

5.1 The alcoholic moiety

5.2 The acyl moiety

5.3 Preparation of pyrroles

6. Experimental

6.1 General Information

6.2 Synthesis of the Compound

6.2.1 3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole

6.2.2 3-(N-tosyl-L-alaninyloxy)-5-phenylthiophene

6.2.3 3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole

6.2.4 3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole

6.3 Preparation and Use of Test Device Containing the Compound

6.3.1 3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole

6.3.2 3-(N-tosyl-L-alaninyloxy)-5-phenylthiophene

6.3.3 3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole

6.3.4 3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole

6.4 Evaluation of the Test Device

1. INTRODUCTION

The present invention relates to novel compounds useful in assaying atest sample for the presence of certain analyte constituents. Suchanalytes include leukocytes, esterase, and protease. The detection ofleukocytes in urine is especially important in medical diagnostics.

The presence of an abnormal level of leukocytes in a patient's urine ispossibly indicative of such pathological conditions as kidney orurogenital tract infection or other dysfunction. Accordingly, accurateurinary leukocyte information can be an invaluable tool to the physicianin diagnosis and treatment of such pathologies.

Traditionally, the medical profession has relied on visual determinationtechniques to count leukocyte population in urine sediment oruncentrifuged urine, a process requiring expensive equipment such as acentrifuge and microscope, as well as inordinate time expenditure on thepart of the clinician. Moreover, the traditional techniques suffer fromthe inadequacy that only intact cells are determined. Leukocytesoccurring in the urinary system are subject to conditions which canfavor extensive cell lysis. For example, it is known that in urines ofabnormally high pH, leukocyte half life can be as low as 60 minutes.Since lysed cells escape detection in visual examination techniques,erroneously low determinations and false negatives can result.

Of the two techniques of microscopic leukocyte analysis--urine sedimentand non-centrifuged, homogenized urine--the former is clearly the mostdesirable. Although dependable results can inure to the latter, urinesediment observation is used in an overwhelming majority of instances.It requires that the urine sample be centrifuged and the sedimentisolated and subjected to microscopic inspection. The analyst thencounts the number of leukocytes appearing in the viewing field. However,this task is further complicated by the presence of other urinarycomponents in the sediment such as epithelial cells and salt particles.The varying content of sediment constituents, coupled with othercomplicating factors including non-homogeneity of the sample anddiffering optical powers among microscope equipment, can lead toenormous errors in the ultimate determination.

It is thus apparent that a quick, facile method of leukocytedetermination, one which would eliminate the need for time-consumingtechniques, as well as cost-consuming equipment, and which would provideaccurate responses to esterase, protease or leukocyte cells, whether thecells were intact or had been lysed, would indeed advance the state ofthe art by a quantum jump. The present invention provides such anadvance. Moreover, it is based, not on the ability to see leukocytes,but on the enzymatic activity they exhibit, and therefore issubstantially free of the inaccuracies described above.

2. BACKGROUND OF THE INVENTION

There exists in the prior art a body of references which disclose theuse of certain esters which, when cleaved by enzymatic activity, resultin the formation of color or other detectable species. Thus, BritishPat. No. 1,128,371 discloses the use of indoxyl and thioindoxyl estersas useful chromogens in detecting hydrolytic enzymes in body fluids. Theenzymes cleave the ester to generate free indoxyl, which subsequentlyoxidizes to form the dimeric product indigo, a readily observable bluedye. Such activity is said to be due to, among other enzymes,cholinesterase. This patent also teaches that, in addition to theindoxyl portion of the ester substrate, the acid radical is chosen withparticular reference to the enzyme to be detected. For example, it isstated that the acid radical can be acetate, or laurate or stearate fordetection of esterase or lipase, respectively. For detecting enzymessuch as phosphatase or sulfatase the acyl radical can be inorganic.Thus, the British Patent can be held to teach the use of chromogenicesters as substrates for determining esterolytic enzymes, such esterscomprising indoxyl or thioindoxyl as the alcoholic moiety of the ester,the acyl moiety being tailored to be conducive to the particular enzymeto be determined.

The effect of careful acyl radical selection is nowhere more clearlyexemplified than in two references which demonstrate esterasespecificity for esters in which the acyl radical comprises anN-protected amino acid or peptide. Thus Janoff, et al., Proc. Soc.Exper. Biol. Med. 136: 1045-1049 (1971) teaches that alanine esters arespecific substrates for esterase obtained from human leukocytes.Specifically this reference teaches that an extract of human leukocytegranules is capable of hydrolyzing N-acetyl-L-alanyl-L-alanyl-L-alaninemethyl ester. Moreover, L-alanine-p-nitrophenol ester was similarlyhydrolyzed to yield the yellow p-nitrophenol colorform.

Similarly, Sweetman et al., Jour. Hist. Soc., 22: 327-339 teaches theuse of 1-naphthyl-N-acetyl-DL-alanine and 1-naphthyl butyrate todemonstrate the presence of esterase, as well as1-naphthyl-N-acetyl-L-alanyl-L-alanine.

U.S. Pat. No. 4,278,763, assigned to Boehringer Mannheim GmbH combinesthese teachings in arriving at the indoxyl or thioindoxyl esters ofamino acids or peptides as still another example of a traditionalchromogenic substrate for leukocytic esterase activity. Moreover theBoehringer patent teaches the equivalence of proteases and esterase intheir esterolytic penchants.

It is known that ester hydrolysis reactions can be activated by thepresence of many nucleophilic agents, including many alcohols. Thus, therate of hydrolysis of phenyl acetate and p-nitrophenyl acetate byesterase is increased 2.5 to 5.5 times upon addition of methanol andbutanol. Greenzaid and Jencks, Biochemistry, 10(7), 12101227 (1971).Moreover, the effect increases with the length of the n-alkyl group.Wynne and Shalatin, Eur. J. Biochem., 31, 554-560 (1972).

In particular, this activation effect of alcohols has been observed withesters of amino acids. p-Nitrophenyl N-acetyl-L-alaninate hydrolysis isactivated (accelerated) by the presence of methanol. Fastrez and Fersht,Biochemistry, 12(11), 2025-2034 (1973). High molecular weight alcoholsincrease the rate of esterase-induced hydrolysis of p-nitrophenylt-BOC-L-tyrosinate. Ashe and Zimmer, Biochem. and Biophys. Res. Comm.,75(1), 194-199 (1977). The disclosure of U.S. Pat. No. 4,299,917describes other known ester hydrolysis activators such as certain metalcomplexes, pyramine derivatives and imidazoles.

Also known is the use of certain diazonium salts to couple with phenolsand pseudophenols to produce azo dyes. Martinet and Dornier Compt.Rend., 170, 592 (1920). Such a technique is used in an esterase analysiswhereby indoxyl acetate is hydrolyzed via esterase to produce indoxyl,which is in turn coupled with a diazonium salt to form the correspondingazo dye. Holt and Hicks, J. Cell Biol. 29, 361-366 (1966); Gossrau,Histochemistry, 57, 323-342 (1978); West German Offenlegungschrift No.31 17 721, filed May 9, 1980.

The dye industry, an art which is clearly not analogous to medicaldiagnostic assays, but which nevertheless has long been a reservoir ofexperimental organic chemistry procedures, provides some guidance as tocertain amino acids and their cyclization reactions. Thus, it is knownto form pyrroles through the addition of glycine to salts ofβ-phenylglycidic acid, followed by treatment with hot acetic anhydride.Madelung and Obermann, Ber., 63, 2870 (1930).

3. SUMMARY OF THE INVENTION

The present invention provides a new compound which is useful indetermining the presence of leukocytes, esterase and/or protease in atest sample. The invention also relates to a method for preparing thecompound. The compound is one having the structure ##STR2## in which: Ais an acid residue,

R is lower alkyl, aryl, carboxyl, carboxyl ester, amido or cyano,

R* is H or lower alkyl, and

X is O, S, or NR', in which R' is H, lower alkyl or aryl.

The presently claimed method relates to preparing the ester (I) whereinthe structure is ##STR3## in which B is an N-blocked amino acid residueor an N-blocked peptide residue, and wherein R is lower alkyl, aryl,carboxyl, carboxyl ester, amido or cyano and R* is H or lower alkyl. Themethod comprises the sequential steps of

(a) forming a 3-hydroxypyrrole having the structure ##STR4## (b) addingan acid halide of a N-blocked amino acid or a N-blocked peptide to the3-hydroxypyrrole in the presence of an organic acid to form a productmixture, and

(c) isolating the ester (II) from the product mixture.

The procedure of step (a) comprises the sequential steps of:

1. causing an aqueous mixture of a ketone, an alkali metalmonopersulfate and a compound having the structure ##STR5## to react inthe presence of a sufficient amount of alkali metal bicarbonate tomaintain the mixture at a pH of at least 7, thereby forming a firstreaction mixture.

2. adding HOOC--CH₂ --NHR', where R' is as defined above, to the firstmixture to form a second reaction mixture;

3. Removing water from the second mixture to form a substantially driedsecond mixture;

4. adding a carboxylic acid anhydride to the dried second mixture in thepresence of an organic base to form a third reaction mixture;

5. subjecting the resultant mixture from step 4 to hydrolyzingconditions to produce a reaction mixture containing a 3-hydroxypyrrole;and

6. isolating the 3-hydroxypyrrole;

4. DEFINITIONS

The following definitions are provided to clarify the scope of thepresent invention, and to enable its formulation and use.

4.1

The expression "acid residue" includes derivative structures ofester-forming acids without their characteristic acidic --OH group.Thus, the term includes the acyl portion of the acids phosphoric,sulfonic, carbonic, carboxylic, and other ester-forming --OHgroup-containing acids.

The terms "N-blocked-amino acid residue" and "N-blocked-peptide residue"require definition on two counts. "N-blocked" refers to the chemistry ofthe amine group of an amino acid or peptide whereby a hydrogen bound tothe nitrogen atom is replaced by a protective group such as acetyl,p-toluenesulfonyl (tosyl) and tert-butyloxycarbonyl (t-BOC) and otherN-protective groups known in the art.

By the expressions "amino acid residue" and "peptide residue" is meantan amino acid or peptide molecule without the --OH of its carboxylgroup.

4.2

By the expression "aryl" is meant any ring system containingaromaticity. Included by the term are such 5- and 6-membered rings aspyrrole, phenyl, and pyridyl, as well as fused ring systems such asnaphthyl. Thus, the aromatic ring system can be heterocyclic orhomocyclic, and can be substituted or unsubstituted, provided thesubstituent group(s) not interfere with the operation or functioning ofthe claimed composition or test device in its capability of detectingleukocyte cells, esterase or protease. Selection of such substituents isa routine laboratory determination, given the present disclosure.

4.3

The expression "lower alkyl", as used in the present disclosure, is analkyl moiety containing about 1-6 carbon atoms. Included in the meaningof lower alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl.sec-butyl, tert-butyl and all isomers of pentyl and hexyl. These can beunsubstituted, or they can be substituted provided that any suchsubstituent group(s) not interfere with the operation or functioning ofthe composition or test device in its capability to detect leukocytecells, esterase or protease. Selection of such substituents is a routinelaboratory determination, given the present disclosure.

5. DETAILED DESCRIPTION OF THE INVENTION

The compound claimed herein includes a broad range of esters, thealcoholic (phenolic) and acyl moieties of which can be chosen to suitparticular needs. Thus, the alcoholic moiety can be tailored to providea particular desired response, such as a particular color or lightabsorbance, and the acyl group can be selected in accordance with aparticular analyte to be detected.

5.1 The Alcoholic Moiety

The alcoholic moiety of the ester is a pseudophenol in that it containsa heteroatom having a pair of electrons which can delocalize to producearomaticity in concert with the double bonds in the ring. Thus theheteroatom X can be oxygen, sulfur or nitrogen. In the case where X isnitrogen, the atom can be unsubstituted i.e., NH, or it can besubstituted with a lower alkyl or aryl group. As a result of thisaromatic character, the compound lends itself easily to coupling with adiazonium salt to form an azo dye, once the ester has become hydrolyzed.

It has been found that various substituents at the 5-position of thering provide enhanced color formation and storage stability. Thus, R canbe lower alkyl, aryl, carboxyl, carboxyl ester, amide, cyano or othersubstituent, provided that any such substituent group not interfere withthe operation or functioning of the composition or test device in itscapability to detect leukocyte cells, esterase or protease. For thepurpose of assaying for leukocytes, esterase or protease, esters inwhich R is phenyl, or p-chlorophenyl are preferred.

5.2 The Acyl Moiety

The acyl moiety A has equally broad scope, and is chosen with theparticular purpose of the assay in mind. Where it is desired to assay atest sample for esterase or protease activity, such as measuringleukocytes in urine, A can be an amino acid or peptide residue.Preferred for such use is N-tosyl-L-alanine residue.

5.3 Preparation of the Pyrroles and Their Esters

The invention also includes a novel method of synthesizing certainpyrrole esters of N-blocked amino acids and peptides (II). The firststep of the procedure comprises the formation of a 3-hydroxypyrrolehaving the structure (III). This is accomplished by a multistepsynthesis beginning with a compound of structure (IV) as the startingmaterial. This compound is reacted in the presence of acetone or otherketone to form an epoxide compound of the structure ##STR6## Theepoxidation is preferably achieved using an alkali metal monopersulfatesuch as KHSO₅ which is available commercially as Oxone® (DuPontCompany). It has been found that pH control in this step¹,2 isdramatically facilitated when the reaction is performed in the presenceof excess alkali metal bicarbonate such as NaHCO₃, i.e., at a pH of atleast 7. Preferably the reaction mixture is kept at a temperature in therange of about 20° to 30° C., although this range is not critical.Acidification of the resultant reaction mixture, followed by theaddition of an organic solvent such as methylene chloride, serves toextract the epoxide into the organic phase.

Next the organic phase is washed with aqueous base. This step rendersthe epoxide water-soluble, thus transferring it to the aqueous phase.The aqueous phase is separated and a glycine added to produce a reactionmixture containing the double salt ##STR7## This step has been found tobe best performed where the aqueous base has a pH range of about 10.5 to12, with about 11.5 being the preferred pH. The temperature for thisstep is preferably elevated to about 100° C., and a substantial amountof low boiling liquid removed (acetone and water). As the vaportemperature approaches 100° C. heating is maintained to continue refluxat or near 100° C. Following cooling, washing with an organic solventsuch as CH₂ Cl₂ and evaporating to dryness (removal of residual water),(VI) is isolated from the residue by boiling in a solvent such asethanol and allowing crystals to separate.

Next, compound (VI) is treated with a carboxylic acid anhydride to form##STR8## where R" is lower alkyl or aryl. Ac is acetate in the casewhere the anhydride is acetic anhydride. This step is conductedinitially at ambient temperature and ultimately heated to about 90° toabout 140° C. A key facet of this step is the inclusion of an organicbase such as pyridine. Preferably Compound (VI) is suspended in thebase, followed by the acid anhydride addition.

The thus-formed reaction mixture containing (VII) can then be evaporatedto dryness to remove residual anhydride and solvent and the product(VII) recovered.

Treatment of (VII) under a hydrolyzing environment leads to theformation of ##STR9## Preferred as a hydrolyzing environment is amixture of an alcohol such as methanol and aqueous base, such as 2NNaOH. It is preferred to mix (VII), the alcohol and aqueous base at alow temperature, such as about -15° C. to about 10° C. Compound (VIII)generally separates out of solution at this temperature and is isolated,such as by filtration, and purified.

The second step in the procedure is the esterification of (VIII) to formthe product ##STR10## in which B is a N-blocked amino acid or peptideresidue. (VIII) is added to an anhydrous solvent, such astetrahydrofuran (THF), diethyl ether and the like, containing an organicbase such as pyridine and an organic acid such as trifluoroacetic,oxalic, citric, acetic or other carboxylic acid. Preferred for this stepare pyridine and trifluoroacetic acid. Next an acyl halide of thedesired blocked amino acid or peptide residue is added slowly. When thereaction is complete it is quenched with water, optionally containing abuffer such as citric acid and ethyl acetate, and the product (IX)recovered.

6. EXPERIMENTAL

The following examples are provided to further assist the reader inmaking and using the present invention. Thus, preferred embodiments aredescribed in experimental detail and analyzed as to their utility. Theexamples are illustrative only, and are in no way intended as limitingthe scope of the invention described and claimed herein.

6.1 General Information

In the following experimental discussion abbreviations are used asindicated:

g=gram

kg=kilogram

L=liter

mL=milliliter

M=molar

mM=millimolar

N=normal

eq=equivalents

mol=gram molecular formula (moles)

mmol=gram molecular formula×10⁻³ (millimoles)

aq=aqueous

hr=hour

TLC=thin layer chromatography

Infrared (IR) spectra were obtained with a Perkin-Elmer Model 710B or237 infrared spectrophotometer as solutions in CHCl₃ unless otherwisenoted; the 1602 cm⁻¹ band of polystyrene film was used as an externalcalibration standard. Signals are reported as cm⁻¹.

Proton magnetic resonance (¹ H NMR) spectra were obtained at 89.55 MHzusing a JEOL FX-900 spectrometer or at 60 MHz using a Varian T-60spectrometer; spectra were obtained in CDCl₃ solution unless otherwisenoted. Chemical shifts are reported in parts per million downfield fromthe internal standard tetramethylsilane.

Carbon-13 magnetic resonance (¹³ C NMR) spectra were obtained at 22.5MHz using a JEOL FX90Q spectrometer with Fourier transform and with fullproton broad-band noise decoupling; spectra were obtained in CDCl₃solution unless otherwise noted. CArbon shifts are reported in parts permillion downfield from the internal standard tetramethylsilane.

Mass spectra (MS) were obtained on a Hewlett-Packard 5985A spectrometeroperating in either an electron impact (EI) or fast atom bombardment(FAB) mode. High-resolution mass spectra were obtained on an AEI MS-902spectrometer.

Organic reagents were obtained from Aldrich Chemical Company and wereused without purification, unless otherwise noted. Inorganic reagentswere ACS reagent grade from Fisher Scientific Company or other majorvendor. Reaction solvents were ACS reagent grade; tetrahydrofuran (THF)was HPLC grade from J. T. Baker Chemical Company. Brine refers to asaturated aqueous sodium chloride solution.

Thin layer chromatograph (TLC) was performed using silica gel 60F-254plates from E. Merck. Column chromatography was performed using E. MerckSilica Gel 60 (70-230 mesh). All melting points and boiling pointsreported are uncorrected.

6.2 Synthesis of the Compound 6.2.1 Synthesis of3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole (4)

The synthesis of (4) is illustrated in the following reaction sequence:##STR11##

N-tosyl-L-alanine

L-alanine (100 g; 1.11 moles) was dissolved in 2.25 L of 1N sodiumhydroxide (aq), cooled to 5° C. and stirred while a solution ofp-toluenesulfonyl chloride (218 g; 1.11 moles) in 450 mL of toluene wasadded slowly. The mixture was stirred at ambient temperature for 20 hr.The layers were separated and the chilled aqueous layer acidified to pH1with concentrated hydrochloric acid. The white solid title compound wascollected by filtration, washed with water and dried. Yield 178.5 g(66%) mp 134°-5° C. IR (CHCl₃) cm⁻¹ 1726, 1340, 1165, 1095; ¹ H NMR(DMSO-D₆) δ 1.20 (d,J=7,3H), 2.40 (s,3H), 3.85 (p,J=8,1H), 6.4 (br d,1H)(CO₂ H), 7.41 (d, J_(AB) =8, 2H) and 7.75 (d, J_(AB) =8,2H) [centerof pattern: 7.58; ΔV_(AB) = 20.49 Hz], 8.03 (br d, J=8,1H)(NH).

N-tosyl-L-alaninyl chloride

Method A

A mixture of N-tosyl-L-alanine (12.4 g; 0.05 mol) and thionyl chloride(25 mL) was heated for 90 minutes at 55° C., and then concentrated onthe rotary evaporator at 40° C. The red solid residue was dissolved in200 mL of boiling CCl₄, decolorized with 20 g of oven dried Norit®211(American Norit Co., Inc.), filtered and chilled. The cream coloredsolid title product was collected by filtration, washed with hexane anddried. Yield 8.48 g (65%) with mp 101°-101.5° C. IR (CHCl₃) cm⁻¹ 3360,3260, 3025, 1775, 1605, 1350, 1170, 910; ^(I) H NMR (CDCl₃) δ 1.48(d,J=7,3H), 2.43 (s, 3H), 4.33 (p,J=8,1H), 6.93 (br d,J=8,1H)(NH), 7.31(d, J_(AB) =8, 2H) and 7.76 (d, J_(AB) =8,2H) [center of pattern: 7.53;ΔV_(AB) = 26.83 Hz].

Anal, calcd. for C₁₀ H₁₂ ClNO₃ S: C,45.89; H,4.62; N,5.35. Found:C,46.63; H,4.90; N,5.19.

Method B

A mixture of N-tosyl-L-alanine (3.1 g; 13 mmol) and thionyl chloride (6mL) was heated for 90 min at 50° C., then diluted with 50 mL of dryhexane. The mixture was stirred rapidly, chilled and the solid productfiltered. Yield 3.15 g (93%) mp 99°-100° C. The IR spectrum wasidentical to that of the recrystallized material prepared by Method A.

2-Hydroxy-3(carboxymethylamino)-hydrocinnamic acid Dipotassium saltdihydrate (1)

A stirred slurry of 1.0 kg of trans-cinnamic acid (6.75 mol) in 4.5 Lacetone was treated first with NaHCO₃ (2.47 kg; 29.4 mol; 4.36 eq) thencarefully with water (4.5 L). The resulting thick mixture was treateddropwise, over 1.5-2.0 hr, with a solution of OXONE monopersulfatecompound (3.78 kg; contains 1.825 eq of KHSO₅) in 0.4 mM aqueousdisodium ethylenediamine tetraacetic acid (EDTA) (14.5 L; prepared bydissolving 2.17 g disodium EDTA dihydrate in 14.5 L distilled water).During this addition the reaction temperature was maintained at 24°-27°C. using a water bath; the reaction pH was noted to be about 7.4. Afterthe addition was completed the mixture was stirred an additional 0.5 hrthen cooled to about 10° C. The reaction was acidified with concentratedHCl (˜1.2 L) to pH=2, while maintaining the temperature at around 10°C., and then treated with CH₂ Cl₂ (5.05 L) and stirred vigorously for 10minutes.

After allowing the mixture to settle, the aqueous layer was decanted andset aside and the organic layer, which contained insoluble salts, wasfiltered through paper with suction. The filtered solids were washedwith CH₂ Cl₂ (1.9 L) and the aqueous layer extracted with this filtrate.The filtered solids were again washed with CH₂ Cl₂ (3.15 L) and theaqueous layer extracted with this filtrate. The combined CH₂ Cl₂ layerswere extracted with a solution of KOH (593.3 g) in water (6.31 L).Gentle heating to about 40° C. is often required to dissolve a solidwhich may separate during the base extraction. The CH₂ Cl₂ layer wasthen extracted with a solution of KOH (99 g) in water (1.5 L) and thecombined base extracts treated with glycine (481.7 g; 6.416 mol; 0.95eq); the organic layer was discarded.

The aqueous solution pH was adjusted to 11.5 with 25% aqueous KOH thenheated to boiling. Approximately 900 mL of low boiling liquid (acetoneand water) was distilled off until the vapor temperature reached 99° C.,following which, the mixture was refluxed for 2 hours. After cooling,the reaction mixture was extracted with CH₂ Cl₂ (3.15 L), the CH₂ Cl₂phase discarded and the aqueous phase evaporated to dryness underreduced pressure with a 70° C. bath. The residue was boiled in 95%ethanol (EtOH) (8.83 L) for 30 minutes, then allowed to cool slowly withstirring, whereupon the product separated as fine crystals. These werefiltered, washed with fresh 95% EtOH (1.26 L) and dried in a 50°-60° C.oven to give the title compound (1.77 kg; 74.6%) as white crystals withmp=120°-2° C. (uncorrected).

IR (KBr) cm⁻¹ 3420 (br.), 1590 (br.), 1410, 1130, 710; ¹ H NMR (D₂O-TSP) δ 3.1 (s, 2H), 3.89 (d, J_(AB) =4,1H) and 4.52 (d, J_(AB) =4,1H)(center of pattern: 4.21; ΔV_(AB) =18.83 Hz.), 4.68 (s, 6H, exchangableprotons), 7.4 (s, 5H); TLC Rf=0.59 (EtOH:1M triethylammoniumbicarbonate, 7:3)

Anal. Calcd. for C₁₁ H₁₅ NO₇ K₂ : C,37.59; H,4.30; N,3.99. Found:C,37.22; H,4.24; N,3.96.

N-acetyl-3-acetoxy-5-phenylpyrrole (2)

A suspension of 2-hydroxy-3-(carboxymethylamino)hydrocinnamic aciddipotassium salt dihydrate (1) (1.0 kg; 2.84 mol) in pyridine (3.0 L)was treated with acetic anhydride (4.0 L) at ambient temperature underan inert gas atmosphere. A mild exothermic reaction ensued and thereaction temperature rose exponentially to 60°-70° C. during a period of1.5-2.5 hours. Once the reaction began to cool the mixture was heated to120°-123° C. for 15 minutes, then allowed to cool to ambient temperatureover 1 hour, during which time pyridinium acetate separated as crystals.The mixture was filtered through paper with suction and the salts washedwith ethyl acetate (EtOAc) until colorless; the filtrate was evaporatedto dryness under vacuum.

The dark red residue was dissolved in EtOAc (3.0 L) washed three timewith water (1.0 L) each), dried over MgSO₄ and treated with Darco®-G60(ICI Americas, Inc.) (300 g). After stirring for 30 minutes the mixturewas filtered through Celite® (Johns-Mannville) and evaporated to drynessunder vacuum to give a reddish-orange oil. This oil was dissolved inwarm 2-propanol (1.2 L), then allowed to cool slowly to ambienttemperature overnight, whereupon a solid separates. The crystallineproduct was filtered, washed with 50% aqueous 2-propanol and dried togive the title compound (417 g; 60%) with mp=58°-60° C. (uncorrected). Aportion was taken up in diethyl ether (Et₂ O), treated with Norit 211,filtered and concentrated under reduced pressure; on standing at 0° C.colorless tiny needles separated. These were filtered, washed with Et₂O:Hexane (1:1) and vacuum dried to give the analytical sample withmp=60°-62.5° C. (uncorrected).

IR (CHCl₃) cm⁻¹ 3020, 1760, 1730, 1595, 1378, 1320, 1220 (br.), 1030,960, 903; ¹ H NMR (CDCl₃) δ 2.23 (s, 3H), 2.27 (s, 3H), 6.18 (d, J=2,1H), 7.35 (s, 5H), 7.42 (d, J=2, 1H); TLC Rf=0.56 (toluene:dioxane,4:1).

Anal. Calcd. for C₁₄ H₁₃ NO₃ : C, 69.12; H, 5.38; N, 5.76. Found: C,68.88; H, 5.25; N, 5.53.

3-Hydroxy-5-phenylpyrrole (3)

A finely divided portion of N-acetyl-3-acetoxy-5-phenylpyrrole (2) (36.8g; 0.15 mol) was freed of oxygen by stirring in a flowing argon streamfor 10 minutes, then suspended in deoxygenated methanol (MeOH) (379 mL),cooled to -6° C. (in a -15° C. methanol (MeOH)/dry-ice bath) under aninert gas atmosphere and rapidly treated with an ice cold deoxygenatedsolution of 2N NaOH (300 mL). The reaction temperature rose immediatelyupon addition of base to 18° C., and after ˜3 minutes the reactionmixture became homogeneous. As the reaction mixture cooled, compound 3separated as fine crystals. After 15 minutes a solution of colddeoxygenated 2M citric acid (150 mL) was added, the resulting mixturewas stirred for 10 minutes, and then filtered. The solid was washedthoroughly with deoxygenated water (200 mL), taking care to minimizeexposure of the product to air, then dried under vacuum overnight toyield the title compound (22.3 g; 93.6%) as light pink tiny needles.

IR (KBr) cm⁻¹ 3400, 3110, 2900, 1600, 1580, 1555, 1480, 1268, 1180, 742,640; ¹ H NMR (DMSO-D⁶) δ6.1 (m, 1H), 6.3 (m, 1H), 7.0-7.7 (m, 5H), 8.0(s, 1H), 10.4 (br s, 1H), TLC Rf=0.20-0.28 (EtOH:CHCl₃, 1:9).

Anal. Calcd. for C₁₀ H₉ NO: C,75.45; H,5.70; N,8.80. Found: C,75.30;H,5.69; N,8.67.

3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole (4)

A solution of anhydrous tetrahydrofuran (THF, 450 mL), pyridine (43.8mL; 0.542 mol; 1.2 eq) and trifluoroacetic acid (85.0 mL; 1.10 mol; 2.4eq), maintained at 0° C. under an inert gas atmosphere, was treated inone portion with 3-hydroxy-5-phenylpyrrole (3) (71.5 g; 0.45 mol; 1.0eq) followed immediately by the dropwise addition, over 5-10 minutes ofa solution of freshly prepared N-toysl-L-alaninyl chloride (141.0 g;0.54 mol; 1.2 eq) in anhydrous THF (450 mL). The resulting mixture wasstirred for 15 minutes at 0° C. The reaction was then quenched byaddition of a solution of 1.0M aqueous citric acid (315 mL) and EtOAc(1.35 L). After brief mixing the phases were separated and the organiclayer washed with a solution of aqueous NaCl (360 mL; 0.18 g NaCl per mLof water). The organic layer was next extracted twice with a solution of5% aqueous NaHCO₃ (1.35 L each), and then washed with another portion ofaqueous NaCl (360 mL; 0.18 g NaCl per mL of water). The reddish brownorganic layer was stirred at ambient temperature for 15 minutes withMgSO₄ (101 g) and Darco-G60 (143 g), then filtered through Celite andevaporated to dryness under vacuum from a 37° C. bath to give (4) as apinkish-white solid. The crude product was ground to a powder anddissolved in warm (50° C.) THF (250 ml), stirred vigorously and dilutedwith n-hexane (250 mL). The stirring was continued for 1 hour at ambienttemperature as the product crystallized. The solid was filtered, washedwith toluene (about 1 L) until the filtrate was colorless, then driedovernight to yield the title compound (112 g; 65%) as a white powderwith mp=154.5°-155° C.

IR (KCl) cm⁻¹ 3350, 3325, 1760, 1508, 1320, 1155, 770; ¹ H NMR (DMSO-d⁶)δ1.33 (d, J=7, 3H), 2.36 (s, 3H), 4.13 (p, J=8, 1H), 6.25 (m, 1H), 6.73(m, 1H), 7.05-7.50 (m, 5H), 7.5-7.85 (m, 4H), 8.42 (d, J=8, 1H), 11.18(br s, 1H); ¹³ C NMR (DMSO-d⁶) δ18.335, 21.001, 51.370, 98.061, 108.336,123.423, 126.024, 126.610, 128.560, 128.756, 129.601, 132.397, 137.600,138.380, 142.737, 169.919; [α]_(D) =-70° (c=1.11, MeOH); TLC Rf=0.45(EtOAc:hexane, 1:1); TLC Rf=0.40 (toluene-dioxane, 4:1).

Anal. Calcd. for C₂₀ H₂₀ N₂ O₄ S: C,62.48; H,5.24; N,7.29. Found:C,62.62; H,5.27; N,7.30.

6.2.2 Synthesis of 3-(N-tosyl-L-alaninyloxy)-5-phenylthiophene (9)

A series of experiments was conducted to prepare3-hydroxy-5-phenylthiophene by minor modifications of the reportedliterature procedures³,4 outlined on the following page. The resultanthydroxythiophene was then acylated with N-tosyl-L-alaninyl chloride togive the corresponding N-tosyl-L-alaninate ester in 46% yield(unoptimized procedure).

3-Phenyl-1,2-dithia-3-cyclopenten-5-one (5)

A suspension of 10 g of ethyl cinnamate (56.82 mmol) and 10 g of sulfurwas heated at 250° C. for four hours in a 50 mL flask equipped with adistillation head and receiver to remove ethanol produced during thereaction. The reaction mixture was then allowed to cool to 100° C. andadded to 500 mL of refluxing ethanol. The resulting precipitate wasfiltered and successively triturated with 500 mL of boiling acetone andtwice with 500 mL portions of ethanol. The combined supernatants wereconcentrated to a black solid, which was crystallized from methanol togive dark brown needles (5). A second recrystallization from methanolusing Norit and filtration through Celite gave 2.023 g of light yellowneedles mp 113°-115° C.

IR (KBr) cm⁻¹ 1650, 1550, 1390, 1350, 1130, 770; ¹ H NMR (60 m Hz,CDCl₃) δ6.92 (s, 1H), 7.58 (m, 5H); TLC Rf=0.5 (dichloromethane).

Anal. Calcd. for C₉ H₆ O₂ S: C,55.64; H,3.11. Found: C,55.53; H,3.47.

cis-4-Keto-6-phenyl-3,7-dithia-5-nonenedioic acid (6)

A molten solution of 35.48 g of sodium sulfide nonahydrate (148 mmol) at94° C. was treated with 6.65 g of3-phenyl-1,2-dithia-3-cyclopenten-5-one (5) (34.23 mmol) addedportionwise over five minutes. After fifteen minutes, the mixture wasadded to a ice-cold solution containing 43.6 g of bromoacetic acid (314mmol) in 60 mL of H₂ O adjusted to pH 8.7 with sodium carbonate. Theresulting solution was maintained at 0° C., pH 8.7 for 45 minutes, andwas then filtered. The filtrate was maintained at 0° C. and acidified topH 3.7 with a 5N HCl solution. The resulting mixture was stirredovernight at 5° C. The supernatant was then decanted, and the resultingoil triturated with ether. The oil was evaporated with toluene until6.98 g of a colorless foam was obtained (65%). This material was usedwithout further purification.

An analytical sample was obtained from the ether supernatant, which uponconcentration, successive evaporation with acetic acid and toluene, andtrituration with ether, gave tan crystals. mp=142.5°-150° C.

IR (KBr) cm⁻¹ 1705, 1655; ¹ H NMR (60 MHz, DMSO-D₆) δ2.06 (s, CH₃ CO₂ Himpurity) 3.30 (s, 2H), 3.77 (s, 2H), 5.67 (m, OH), 6.37 (s, 1H), 7.43(m, 5H); TLC Rf=0.85 (chloroform:methanol:acetic acid, 5:5:1).

Anal. Calcd. for C₁₃ H₁₂ S₂ O₅ : C, 50.00; H, 3.88. Found: C, 50.26; H,3.98.

3-Hydroxy-5-phenylthiophene Acetate (7)

A vigorously stirred suspension of 3.40 g of crudecis-4-keto-6-phenyl-3,7-dithia-5-nonenedioic acid (6) (10.9 mmol), 3.40g of sodium acetate (41.5 mmol), and 30 mL of acetic anhydride washeated to refluxe for one hour. The mixture was allowed to cool and wasthen filtered and evaporated to give a black oil. This residue wasdissolved in 75 mL of ethyl acetate and extracted three times with a 50mL portion of ice-cold saturated sodium bicarbonate solution. Theorganic layer was then washed with brine, dried over sodium sulfate,filtered, and evaporated to give 2.826 g of a black solid. The crudeproduct (7) was purified by evaporative distillation at 120°-140° C. and0.1 mm to give 1.235 g of a light orange oil which solidified uponstanding (52%).

IR cm⁻¹ 1700, 1745; ¹ H NMR (60 MHz, CDCl₃) δ2.23 (S, 3H), 7.03 (d,J=2Hz, 1H), 7.13 (d,J=2 Hz, 1H); 7.23-7.73 (m, 5H); MS (EI, DIP) m/e 218(M⁺, 12.6%); TLC Rf=0.48 (hexane:ethyl acetate, 5:1).

Anal. Calc. for C₁₂ H₁₀ SO₂.1/2H₂ O: C, 63.41; H, 4.88. Found: C, 63.78;H, 4.86.

3-Hydroxy-5-phenylthiophene (8)

A mixture of 2.126 g of 3-hydroxy-5-phenylthiophene acetate (7) (9.74mmol) and 80 mL of methanol under an argon atmosphere was treated with11 mL of 1N NaOH. After 20 minutes, the reaction was quenched by theaddition of 11 mL of 1N HCL, evaporated at 25° C., 12 mm Hg, toapproximately 50 mL volume, and treated with 100 mL of ethyl acetate.The organic layer was separated, washed with brine, dried over sodiumsulfate, filtered, and evaporated to give a black solid. This residuewas dissolved in 75 mL of ethyl acetate and dried over MgSO₄. Filtrationand evaporation gave a black solid which was triturated four times withhot hexane to give upon cooling a total of 837 mg of a yellow solid (8),mp 74°-75° C. (49%). The combined mother liquors were concentrated togive 0.87 g of a solid which was chromatographed over 100 g of SiO₂eluted with a hexane:ethyl acetate (7:1) solvent mixture. Obtained afterrecrystallization was an additional 380 mg of product. mp 73.5°-74° C.The combined yield was thus 1.217 g (71%). mp 74.5°-75° C. (Lit³,4 75°C., 78° C.).

IR cm⁻¹ 3380, 1635; ¹ H NMR (90 MHz, CDCl₃) δ3.81 (s, 2H), 6.57 (s, 1H),7.2-7.7 (m, 5H); MS (EI) m/e 176.0 (70.7%); TLC Rf=0.23 (hexane:ethylacetate, 1:5).

Anal. Calcd. for C₁₀ H₈ OS: C, 68.15; H, 4.57. Found: C, 68.05; H, 4.70.

3-(N-tosyl-L-alaninyloxy)-5-phenylthiophene (9)

A solution containing 440 mg of 3-hydroxy-5-phenylthiophene (8) (2.5mmol) in 20 mL of dichloromethane and 0.61 mL of pyridine (7.5 mmol) at0° C. under an argon atmosphere was treated with a solution containing1.314 g of N-tosylalaninyl chloride (5 mmol) in 10 mL of dichloromethaneadded dropwise over a period of five minutes. The reaction was allowedto stir for 0.5 hour at 0° C., and was then poured into 100 mL ofchloroform. The mixture was then successively extrated with 50 mLportions of 1N citric acid, water, ice-cold sodium bicarbonate solution,water, and brine. The mixture was then dried over sodium sulfate,filtered, and evaporated to give 1.78 g of a brown oil. Attemptedcrystallization from toluene after treatment with 1.78 g of Norit wasunsuccessful. The residue was then chromatographed on a 200 g column ofSiO₂ eluted with dichloromethane at a flow rate of 10 mL/minute.Fractions containing the product were pooled and concentrated to give951 mg of a reddish oil. The product was crystallized from toluene.Successive recrystallizations from toluene gave a total of 463 mg ofproduct (9) as light yellow solid, (46%). mp 85°-87° C.

IR (KCl) cm⁻¹ 1735, 1330, 1150; ¹ H NMR (90 MHZ, CDCl₃) δ1.53 (d, J=7Hz, 3H), 1.62 (s, 3H), 4.23 (m, 1H), 5.32 (d, J=9 Hz, 1H), 6.84 (d,J=1.4 Hz, 1H), 6.88 (d, J=1.4 Hz, 1H), 7.23-7.83 (m, 9H); MS (FAB) m/e402 (M+1, 15%); TLC Rf=0.20 (hexane:ethyl acetate, 4:1).

Anal. Calcd. for C₂₀ H₁₉ NO₄ S: C, 59.83; H, 4.77; N, 3.59. Found: C,59.60; H, 4.77; N, 3.43.

6.2.3 Synthesis of 3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole(13)

A series of experiments was conducted to prepare the captioned estercorresponding to compound (I) in which A is N-tosyl-L-alaninyl, R isphenyl, R* is H, X is NR' and R' is CH₃. The reaction sequence is asfollows: ##STR13##

2-Hydroxy-3-(N-methylcarboxymethylamino)-hydrocinnamic acid dipotassiumsalt (10)

A mixture of β-phenylglycidic acid potassium salt (30 g; 0.15 mole),N-methylglycine (13.2 g; 0.15 mole), distilled water (119 ml) and KOHsolution (9N; 22.3 ml) was heated to reflux for 3 hours to give a lightyellow solution. The reaction mixture was evaporated to dryness underreduced pressure at 70° C. The residue was then crystallized from 95%EtOH (100 ml) to give a white solid which, after drying overnight underreduced pressure at 110° C., yielded 30.8 g of white solid (10) (yield63%).

IR (KCl) cm⁻¹ 3360 (br.), 1580, 1405, 705; ¹ H NMR (CD₃ OD) δ2.30 (s,3H), 2.98 (s, 2H), 3.70 (d, J=3 Hz, 1H), 4.48 (d, J=3 Hz, 1H), 4.92 (s,1H), 7.40 (s, 5H); TLC Rf=0.51 (EtOH:1M triethylammonium bicarbonate,7:3). (Product had no melting point less than 270° C.).

3-Acetoxy-1-methyl-5-phenylpyrrole (11)

A mixture of 2-hydroxy-3-(N-methylcarboxymethylamino)-hydrocinnamic aciddipotassium salt (10) (15.2 g, 46 mmole), acetic anhydride (173 ml) andtriethylamine (308 ml) was heated at 90° C. for 19 hrs. The reactionmixture, which became deep brown in color, was filtered and the solidwashed with ether. The filtrate was evaporated under reduced pressure togive a deep brown residue, which was taken up in ether (300 ml) andwater (200 ml). The layers were separated and the ether layer washedwith another portion of water (200 ml). The ether solution was thendried over MgSO₄, filtered and concentrated under reduced pressure togive 10.7 g of brown residue which after Kugelrohr distillation andcrystallization from ether yielded 3.0 g of white crystals (11) (yield30%) mp=64°-65° C.

IR (CHCL₃) cm⁻¹ 2990, 1750, 1570, 1518, 1482, 1375, 1240 (br.), 1024,910, 700; ¹ H NMR (CDCl₃) δ2.20 (s, 3H), 3.58 (s, 3H), 6.10 (d, J=2 Hz,1H), 6.75 (d, J=2 Hz, 1H), 7.35 (s, 5H); TLC Rf=0.58 (hexane-EtOAc 7:3)

Anal. Calcd. for C₁₃ H₁₃ NO₂ : C, 72.54; H, 6.10; N, 6.44. Found: C,72.57; H, 6.09; N, 6.51.

3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole (13)

To a mixture of deoxygenated methanol (15.5 ml) and3-acetoxy-1-methyl-5-phenylpyrrole (11) (1.3 g, 6.2 mmole), under argon,was added deoxygenated NaOH (2N, 12.5 ml). The reaction mixture wasstirred in an ice-bath for 15 minutes. Then deoxygenated citric acid(2M, 7 ml) was added and the resulting mixture was stirred in an icebath for 8 minutes. The reaction mixture was concentrated under reducedpressure, then 20 ml of water was added and was extracted twice withethylacetate (EtOAc) (50 ml). The EtOAc layers were combined, dried overMgSO₄, filtered and concentrated under reduced pressure to give3-hydroxy-1-methyl-5-phenylpyrrole (12) as an orange residue. Underargon, a cold solution of anhydrous THF (12.4 ml), pyridine (0.6 ml, 7.4mmole, 1.2 eq) and trifluoroacetic acid (1.2 ml, 15 mmole, 2.4 eq) wasadded to the orange residue, followed immediately by the additon of asolution of freshly prepared N-tosyl-L-alaninyl chloride (1.2 g, 7.4mmole, 1.2 eq) in anhydrous THF (12.4 ml). The resulting mixture wasstirred for one hr at 0° C. Then the reaction was quenched by theaddition of aqueous citric acid (1M, 5 ml) and EtOAc (30 ml). After abrief mixing, the layers were separated and the organic layer wassuccessively washed with saturated NaCl solution, twice with 5% NaHCO₃solution and again with saturated NaCl solution. The EtOAc extract wasthen dried over MgSO₄, treated with Norit 211, filtered and concentratedunder reduced pressure to give the crude product (13) as an orangeresidue. This was dissolved in hexane:EtOAc (1:1) (5 ml) andchromatographed on a column (SiO₂, 100 g) by elution with hexane:EtOAc(7:3) to give 1 g of (13) as a thick light orange oil. A portion of thiscrude product was further purified by semi-preparative HPLC (column, IBMsilica, 1 cm×25 cm; mobile phase, hexane:EtOAc 8:2; flow rate, 4.0ml/min; pressure, 0.2 psi) to yield a honey color thick oil (13).

IR (film) cm⁻¹ 3260, 2950, 1760, 1520, 1350, 1170, 770; ¹ H NMR(DMSO-d₆) δ1.28 (d, J=7 Hz, 3H), 2.36 (s, 3H), 3.58 (s, 3H), 5.85 (d,J=2 Hz, 1H), 6.15 (m, 1H), 6.74 (d, J=2 Hz, 1H), 7.30-780 (m, 9H), 8.37(d, J=8 Hz, 1H); ¹³ C NMR (DMSO-d₆) ppm 8.205, 20.936, 34.917, 51.240,100.598, 113.148, 126.544, 127.000, 128.105, 128.560, 129.601, 130.901,132.202, 135.714, 138.315, 142.672, 169.724; TLC Rf=0.52(toluene:dioxane 4:1); High-resolution mass spectrum, C₂₁ H₂₂ N₂ O₄ Srequires m/e 398.1300, found m/e 398.1297.

6.2.4 Synthesis of 3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole(18)

A series of experiments was conducted to prepare the captioned estercompound corresponding to compound (I) in which A is N-tosyl-L-alaninyl,R is p-chlorophenyl, R* is H, X is NR' and R' is H. The reactionsequence is as follows: ##STR14##

trans-β-(p-Chlorophenyl)glycidic acid (14)

To a stirred slurry of p-chlorocinnamic acid (68.5 g; 0.375 mol) in 260mL of acetone was added NaHCO₃ (137 g; 1.63 mol), followed by slowaddition of 260 mL of water. To this mixture was added, over 2.5 hoursat 22°-27° C., a mixture of OXONE (211 g; 0.343 mol), 120 mg of disodiumEDTA and 805 mL of water. After five hours the mixture was acidifiedwith 70 mL of cold 12N HCL, to bring the pH down to about 2.5, and itthen was extracted with 700 mL of ethyl acetate. The extract was washedwith brine, dried with MgSO₄, filtered, and the filtrate was evaporatedto dryness under vacuum. The white solids were crystallized from ethylacetate: mp 121°-5° C. (72.2 g; 97% yield). ¹ H NMR (CDCl₃ /DMSO-D₆)δ7.3 (m, 4H), 4.05 (d, J= 2, 1H), and 3.4 (d, J=2, 1H).

Anal. Calcd. for C₉ H₇ ClO₃ : C, 54.43; H, 3.55; Cl, 17.85. Found: C,54.53; H, 3.80; Cl, 17.91.

2-Hydroxy-3-(carboxymethylamino)-p-chlorohydrocinnamic acid dipotassiumsalt dihydrate (15)

To a solution of KOH (85%) (46.7 g; 0.709 mol) and 400 mL of water wasadded glycine (25.9 g; 0.345 mol) followed bytrans-β-p-chlorophenylglycidic acid (14) (72.2 g; 0.3635 mol). Thismixture was heated at 100° C. for two hours, cooled to room temperatureand sufficient KOH added to raise the pH to 12. The turbid solution wasextracted three times with ethyl acetate, which extract was thendiscarded; the clear aqueous solution (about 500 mL) was evaporatedunder vacuum to dryness using a 70° water bath. The solids were thandissolved in about 350 mL of hot ethanol, filtered, and the filtratechilled in an ice bath for several hours. The crystallized solids werecollected by filtration and washed with some cold ethanol: mp 93°-5° C.with decarboxylation at 185° C. (57.2 g; 41%).

¹ H NMR (D₂ O-TSP) δ7.4 (s, 4H), 4.4 (d, J=4, 1H), 4.05 (d, J=4, 1H),and 3.1 (s, 2H).

Anal. Calcd. for C₁₁ H₁₀ ClNO₅ K₂.2H₂ O: C, 34.24; H, 3.66; N, 3.63.Found C, 34.40; H, 4.03; N, 3.42.

N-Acetyl-3-acetoxy-5-(p-chlorophenyl)pyrrole (16)

To the 2-hydroxy-3-(carboxymethylamino)-p-chlorohydrocinnamic aciddipotassium salt dihydrate (15) (10 g; 0.02591 mol) was added aceticanhydride (40 mL) and pyridine (30 mL). This mixture was gently heatedto 35° C. at which point the solution exothermed to 67° then began tofall, whereupon heating was again resumed. The mixture was heated at121°-2° C. (internal temperature) for one hour then cooled. To thereaction mixture was added about 30 mL of ethyl acetate whichprecipitated most the pyridinium acetate salt; this salt was collectedby filtration and washed with a small amount of ethyl acetate. Thefiltrate was then evaporated under vacuum to an oil and ice water added.The product was extracted with ether and the ether extracts weresuccessively washed twice with cold dilute aqueous citric acid, coldwater, three times with cold dilute aq. NaHCO₃, cold water and brine,followed by drying over MgSO₄ and filtering. The filtrate was treatedwith 10 g of Darco, stirred for 20 minutes and then filtered. Thefiltrate was evaporated under vacuum to an oil. To the oil was added 25mL of 2-propanol. The resultant solution yielded, with chilling andscratching, pale yellow crystals: mp 69°-71° C. (3.4 g; 47%); TLCRf=0.61 (toluene:dioxane, 95:5). An analytical sample was recrystallizedfrom 2-propanol but no change in mp was observed.

IR (KCl) cm⁻¹ 1755 (C═O, ester) and 1730 (C═O, amide); ¹ H NMR (CDCl₃)δ7.4 (m, 5H), 6.2 (d, J=2, 1H), 2.4 (s, 3H) and 2.3 (s, 3H).

Anal. Calcd. for C₁₄ H₁₂ ClNO₃ : C, 60.55; H, 4.36; N, 5.04. Found: C,60.65; H, 4.55; N, 5.07.

3-Hydroxy-5-(p-chlorophenyl)pyrrole (17)

A sample of N-acetyl-3-acetoxyl-5-p-chlorophenylpyrrole (16) (2.8 g;0.01 mol) was deoxygenated for ten minutes with a stream of N₂. Thesolids were then dissolved in deoxygenated methanol (30 mL) which wasthen chilled to -8° C. At once was added a cold deoxygenated solution ofNaOH (1.6 g; 0.04 mol) in 20 mL H₂ O, which solution was then heatedbriefly to 15° C. and then immediately cooled to -5° C.; after 25minutes the clear solution was treated with a cold deoxygenated solutionof citric acid (4.2 g; 0.02 mol) in 15 mL H₂ O. The temperature rosebriefly to 5° C. After 0.5 hr. stirring at -5° C., the solids werecollected by filtration and washed with cold deoxygenated H₂ O. The palegreen product was dried under vacuum at room temperature over P₂ O₅ forseveral days (1.3 g; 68%); TLC Rf=0.19 (CHCl₃ :EtOH, 9:1); IR (KCl)showed no evidence for C═O absorption.

Anal. Calcd. for C₁₀ H₈ ClNO.1/6H₂ O: C, 61.08; H, 4.27; N, 7.12. Found:C, 61.36; H, 4.44; N, 6.85.

3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole (18)

To N₂ deoxygenated THF (15 mL) was added pyridine (0.65 mL; 0.008 mol),trifluroracetic acid (1.27 mL; 0.0164 mol), and3-hydroxy-5-(p-chlorophenyl)pyrrole (17) (1.3 g; 0.0065 mol). Thesolution was chilled to 0° C. to -4° C. and a N₂ deoxygenated chilled(0° to -4° C.) solution of N-tosyl-L-alaninyl chloride (2.1 g; 0.008mol) in 15 mL of THF was added over 10 minutes. After maintaining themixture at 0° C. for one hour, a mixture of ice and 100 mL of 1N citricacid was added. This mixture was extracted with ethyl acetate and theextract washed once with cold brine, twice with cold dilute NaHCO₃, andonce with cold brine, following which, it was dried over MgSO₄ andfiltered. The filtrate was treated with 2 g of Darco and stirred for tenminutes, filtered and the filtrate concentrated under vacuum to areddish-brown oil. A second treatment with 1.3 g Darco afforded a lightreddish oil. The oil was dissolved in toluene:cyclohexane (4:1) andplaced in the refrigerator overnight. Light salmon crystals wereobtained. (1.45 g; 53%); mp 113°-5° C.; TLC Rf=0.47 (Et₂ O); IR (KCl)cm⁻¹ 1740 (C═O, ester); ¹ H NMR (CDCl₃) δ 8.4 (br s, 1H), 7.8-7.2 (m,8H), 6.7 (m, 1H), 6.2 (m, 1H), 5.5 (d, J=9, 1H), 4.2 (p, J=8, 1H), 2.4(s, 3H), 1.4 (d, 3H); MS (EI, DIP) m/e 418 (M⁺, 2.3%) and 420 (M⁺,0.8%).

Anal. Calcd. for C₂₀ H₁₉ ClN₂ O₄ S: C, 57.34; H, 4.57; N, 6.69. Found:C, 57.53; H, 4.58; N, 6.67.

6.3 Preparation and Use of Test Devices Containing the Compound

A series of experiments was conducted to prepare test devices containingthe present invention in which the ester substrates of paragraph 7.1,supra, were tested for responsiveness to leukocytes in urine. Thedevices comprised a small square of filter paper containing the assayreagents, the paper mounted at one end of a polystyrene film strip. Thefilter paper was impregnated with buffer, the ester an accelerator and adiazonium salt coupling agent. Each of the devices tested was found toexhibit a positive test for leukocytes in urine.

6.31 Test device in which the ester is3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole (4)

A test device, sensitive to the presence of leukocytes in urine, wasprepared. The device comprised a small square of filter paper mounted atone end of an oblong strip of polystyrene film. The paper wasimpregnated with various ingredients including a chromogenic ester, anaccelerator and a diazonium salt. A 2 inch wide strip of Eaton andDickman #205 filter paper was immersed in an aqueous solution containingthe following:

0.4M borate-NaOH buffer pH=8.6

2.0% (w/v) polyvinylpyrrolidone K-30

0.2% (w/v) Bioterge AS-40

0.25M NaCl

The paper was then dried for 7 minutes in an Overly Air Foil paper dryerat 175°-200° F. at an airflow pressure of 1 inch of water. Next, thedried paper was immersed in an acetone solution containing

2.0% (v/v) n-decanol

0.75 mM 2-methoxy-4-morpholinobenzene diazonium chloride

0.5 mM 3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole

Following this impregnation the paper was dried for 10 minutes in aventilated Hotpack® oven at 130° F. An off-white test paper wasobtained.

A piece of the dried, impregnated paper was cut to a square measuring0.2 inches on a side and mounted at one end of an axially orientedpolystyrene strip measuring 4 inches by 0.2 inches. Mounting the paperto the strip was achieved using Double Stick® double faced adhesive (3MCompany).

6.3.2 Test device in which the ester is3-(N-tosyl-L-alaninyloxy)-5-phenylthiophene (9)

A test device sensitive to the presence of leukocytes in urine wasprepared, wherein 3-(N-tosyl-L-alaninyloxy)-5-phenylthiophene was usedas the indicator. A piece of filter paper (Eaton & Dikeman #205) wasimmersed in an aqueous first solution containing the following:

0.4M borate-NaOH buffer (pH=8.5)

0.4M NaCl

1.5% (w/v) polyvinylpyrrolidone (K-30)

The impregnated paper was dried in a forced air oven for 30 minutes at70° C., whereupon it was permitted to cool to room temperature andimpregnated with a second dip solution comprising an acetone solutioncontaining:

0.75 mM 3-(N-tosyl-LL-alaninyloxy)-5-phenylthiophene

0.75 mM 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloridedouble salt

0.5% (v/v) n-decanol

The doubly impregnated paper was then dried in a forced air oven for 5minutes at 50° C.

The dried paper was cut into squares measuring 0.2 inches on a side andmounted at the end of polystyrene film strips measuring 0.2 by 3.25inches. Mounting was accomplished using Double Stick, a double facedadhesive from 3M Company. The test devices were stored in bottles of 100each, together with silica gel and molecular sieves to providedessication.

6.3.3 Test device in which the ester is3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole (13)

Test devices were prepared following the procedure in experiment 6.3.1in which the ester indicator was3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole and the couplingagent was 1-diazonaphthalene-4-sulfonate. The aqueous first dip solutioncontained:

0.4M boric acid

2.0% (w/v) polyvinylpyrrolidone (K-30)

0.2% (v/v) Bioterg AS-40

0.25M NaCl

Prior to impregnation of the filter paper, the solution was titratedwith NaOH to a pH of 9.0.

The second dip solution in acetone contained:

0.75 mM 1-diazo-2-naphthol-4-sulfonate

1.3 mM 3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenylpyrrole

1.5% (v/v) dodecanol

Following impregnation in the aqueous first dip, the paper was dried forabout 5 minutes at about 80° C., and for about 5 minutes at 70° C.following impregnation in the acetonic second dip.

The dried paper was mounted as in experiment 6.3.1.

6.3.4 Test device in which the ester is3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole (18)

Test devices were prepared as in Experiment 6.3.1 except that theacetone solution contained, in place of the phenylpyrrole, 1.3 mM3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole.

6.4 Evaluation of the Test Device

The test devices prepared in the above experiments were subjected toevaluation of their ability to detect leukocytes present in urine.

Test samples were prepared from a normal human urine pool. One sampleserved as a blank and leukocytes isolated from freshly drawn blood wereadded to two additional urine samples to yield concentrations of 0, 10and 75 leukocytes/μL, respectively.

Test devices were quickly immersed in and removed from a test sample.Two minutes later the devices were observed using a spectrophotometer tomeasure % reflectance at different wavelengths from 400-700 nm(nanometers).

The data show that all of the test devices demonstrated clearlydiscernable differences in light reflectance corresponding to differentleukocyte levels in the test samples. The data are presented in thefollowing table.

    ______________________________________                                                     Leukocyte                                                                     Concentration                                                                             % Reflectance                                        Experiment No.                                                                             (cells/μL)                                                                             at 555 nm                                            ______________________________________                                         6.3.1*       0          --                                                                10-12       --                                                   6.3.2         0          65                                                                10          60                                                                65          42                                                   6.3.3         0          67                                                                10          64                                                                75          60                                                   6.3.4         0          61                                                                10          51                                                                75          42                                                   ______________________________________                                         *Visual observation: purple color formed at 10-12 cells/μL; blank gave     no color change                                                          

What is claimed is:
 1. A compound having the structure ##STR15## inwhich: A is --COCH₃ or ##STR16## where Ts is tosyl; R is a lower alkylgroup having 1 to 6 carbon atoms, phenyl or chlorophenyl;R* is H or alower alkyl group having 1 to 6 carbon atoms; and X is NR', in which R'is H or a lower alkyl group having 1 to 6 carbon atoms.
 2. The compound3-(N-tosyl-L-alaninyloxy)-5-phenyl pyrrole.
 3. The compound3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenyl pyrrole.
 4. The compound3-acetoxy-1-methyl-5-phenyl pyrrole.
 5. The compound3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole.